Malaria control and eradication strategies continue to primarily rely on vector control that is stymied by insecticide resistance in vector populations. In addition to resistance management, novel insecticides with new modes of action - particularly for which efficacy is not affected by existing resistance mechanisms - are required. The long-term goal is to develop a malaria vector control methodology based on a new insecticide target, the serine protease inhibitor serpin (SRPN)2 from the African malaria mosquito, Anopheles gambiae s.s.. SRPN2 is a key negative regulator in the extracellular proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization - a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity with escalating daily mortality nine days after treatment. The objectives in this application are to identify the interactions of SRPN2 with its proteinase targets and to evaluate the malaria control potential of a SRPN2 inhibitor. The rationale for the proposed research is that detailed information on SRPN2's biological proteinase targets and mode of action will ultimately allow the design of small molecule inhibitors of SRPN2 that could act as an insecticide for mosquito vector control. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the molecular targets of SRPN2 inhibitory function; (2) Determine the molecular interface between SRPN2 and its target proteinase(s); and (3) Model the impact of SRPN2 depletion on disease transmission. Under the first aim, a combination of biochemical and genetic approaches that have been used successfully in the applicant's laboratory will be used to identify serine proteinase targets of SRPN2 based on their ability to (a) form covalent complexes with the serpin and (b) revert the serpin's depletion phenotype. Under the second aim, the molecular interactions between SRPN2 and one of its target proteinases, CLIPB9, will be identified by protein crystallography and mutational analysis. Under the third aim, the potential effect of SRPN2 inhibition on malaria transmission and resistance development against a potential SRPN2 inhibitor will be assessed by mathematical modeling. The proposed research is innovative, as it will for the first time evaluate the mosquito immune system as a physiological target for novel insecticides. Additionally, this project will use microfluidics as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect biochemistry. This project is significant as it will provide fundamental knowledge of the nature of serine proteinase cascades that regulate melanization in mosquitoes. Ultimately, it has the potential to advance the development of new a generation of insecticide targets for malaria control.